betadex and Leishmaniasis--Visceral

2-n-propylquinoline (2-n-PQ) had shown interesting in vivo antileishmanial activities after administration by oral route on leishmaniasis animal models. However, the lipophilic properties of this compound avoid its use by intravenous route, this route being indicated in cases of severe visceral leishmaniasis with vomiting. Thus, a 2-n-propylquinoline hydroxypropyl beta-cyclodextrin (2-n-PQ-HPC) formulation was set up in this aim. The formulation was active in vitro both on Leishmania donovani axenic and intramacrophage amastigotes with IC50 values at 6.22±0.82μM and 20.01±0.52μM, respectively, without any toxicity on macrophages. 2-n-PQ-HPC exhibited similar activity on WT and drug-resistant parasites. Its in vitro interactions with antimonials, amphotericin B and miltefosine were found as additive both in axenic amastigotes and intramacrophage amastigotes. 2-n-PQ-HPC was not able to generate drug resistance after in vitro drug pressure since the resistance index was less than 4. 2-n-PQ-HPC was also active on the L. donovani/Balb/c mice model with an intravenous treatment regimen at 10mgkg(-1)day(-1) on 10 consecutive days without hepatic, renal and blood toxicity. The pharmacokinetics of 2-n-PQ in rats showed that after an intravenous treatment of the formulation at 10mgkg(-1), the plasma drug concentrations rapidly declined bi-exponentially with a half-life of 58.7min and a total clearance of 18.63lh(-1)kg(-1). The apparent volume of distribution was higher than the blood volume in rats, indicating that 2-n-PQ was well distributed in tissues, allowing parasite elimination. Such a formulation is worth of further antiparasitic and toxicological evaluations.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Administration, Oral; Animals; Antiprotozoal Agents; beta-Cyclodextrins; Disease Models, Animal; Drug Resistance; Drug Therapy, Combination; Female; Half-Life; Inhibitory Concentration 50; Leishmania donovani; Leishmaniasis, Visceral; Male; Mice; Mice, Inbred BALB C; Quinolines; Rats; Rats, Sprague-Dawley; Tissue Distribution

The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins.

Topics: Animals; beta-Cyclodextrins; Cell Membrane; Complement Activation; Complement C3b; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Female; Gas Chromatography-Mass Spectrometry; Glycosylphosphatidylinositols; Humans; Leishmania infantum; Leishmaniasis, Visceral; Macrophages; Mass Spectrometry; Mice; Mice, Inbred BALB C; Parasite Load; Parasitic Sensitivity Tests; Protein Binding; Proteolysis; Protozoan Proteins; Serum; Sterols; Virulence Factors

Pentamidine isethionate (PNT) is an antiprotozoal active in many cases of leishmaniasis, despite the present limitations including high toxicity and parenteral administration. In the present work, a PNT encapsulation strategy into β-cyclodextrin cavity at 1:1 and 2:1 (βCD:PNT) molar ratios was used in order to improve the drug's physical and chemical properties. Combining thermodynamic and structural approaches such as isothermal titration calorimetry (ITC), electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance ((1)H NMR, and ROESY) the inclusion process and the thermodynamics parameters were identified. ITC and ESI-MS experimental data suggest the simultaneous formation of different supramolecular complexes in solution. Moreover, NMR data are in accordance with these results, suggesting a deep inclusion of PNT into the βCD cavity, through correlations observed in 2D ROESY contour maps. The systems were also characterized by FTIR, TG/DTA and SEM. These techniques indicate the formation of inclusion complex in the solid state. In vivo PNT activity was evaluated orally in mice. The inclusion complex showed a significant reduction of parasite load compared to free PNT.

Topics: Animals; Antiprotozoal Agents; beta-Cyclodextrins; Female; Leishmania infantum; Leishmaniasis, Visceral; Mice; Mice, Inbred BALB C; Parasite Load; Pentamidine; Solubility; Water

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X(1-5)-Y-X(1-5)-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (K(D): 4.27×10(-9) M versus 2.69×10(-7) M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol.

Topics: Animals; beta-Cyclodextrins; Cell Line, Tumor; Cholesterol; Gene Expression Regulation; Genes, Reporter; Glycosphingolipids; Interferon gamma Receptor; Interferon-gamma; Janus Kinase 1; Janus Kinase 2; Leishmania donovani; Leishmaniasis, Visceral; Macrophages; Membrane Microdomains; Mice; Mice, Inbred BALB C; Parasite Load; Phosphorylation; Receptors, Interferon; Signal Transduction; STAT1 Transcription Factor; Transfection

Leishmania are obligate intracellular parasites that invade and survive within host macrophages and can result in visceral leishmaniasis, a major public health problem worldwide. The entry of intracellular parasites, in general, involves interaction with the plasma membrane of host cells. Cholesterol in host cell membranes was recently shown to be necessary for binding and internalization of Leishmania and for the efficient presentation of leishmanial antigens in infected macrophages. This article describes the need to explore cyclodextrin-based compounds, which modulate host membrane cholesterol levels, as a possible therapeutic strategy against leishmaniasis in addition to other intracellular parasites.

Topics: Animals; beta-Cyclodextrins; Cholesterol; Humans; Leishmania donovani; Leishmaniasis, Visceral; Membrane Microdomains

Leishmania donovani is an obligate intracellular parasite that infects macrophages of the vertebrate host, resulting in visceral leishmaniasis in humans, which is usually fatal if untreated. The molecular mechanisms involved in host-parasite interaction leading to attachment on the cell surface and subsequent internalization of the parasite are poorly characterized. Cholesterol is a major constituent of eukaryotic membranes and plays a crucial role in cellular membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains in membranes. Recent observations suggest that cholesterol exerts many of its actions by maintaining a specialized type of membrane domain, termed "lipid rafts", in a functional state. Lipid rafts are enriched in cholesterol and sphingolipids, and have been thought to act as platforms through which signal transduction events are coordinated and pathogens gain entry to infect host cells. We report here that cholesterol depletion from macrophage plasma membranes using methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction in the extent of leishmanial infection. Furthermore, the reduction in the ability of the parasite to infect host macrophages can be reversed upon replenishment of cell membrane cholesterol. Interestingly, these effects were not observed when parasites were serum-opsonized, indicating a specific requirement of cholesterol to mediate entry via the non-opsonic pathway. Importantly, we show that entry of Escherichia coli remains unaffected by cholesterol depletion. Our results therefore point to the specific requirement of plasma membrane cholesterol in efficient attachment and internalization of the parasite to macrophage cells leading to a productive infection. More importantly, these results are significant in developing novel therapeutic strategies to tackle leishmaniasis.

Topics: Animals; beta-Cyclodextrins; Cell Adhesion; Cell Line; Cell Membrane; Cholesterol; Cyclodextrins; Cytoplasm; Leishmania donovani; Leishmaniasis, Visceral; Macrophages; Membrane Microdomains; Mice